Microcystin-LR Induces Estrogenic Effects at Environmentally Relevant Concentration in Black-Spotted Pond Frogs (Pelophylax nigromaculatus): In Situ, In Vivo, In Vitro, and In Silico Investigations.

Harmful cyanobacterial blooms are frequent and intense worldwide, creating hazards for aquatic biodiversity. The potential estrogen-like effect of Microcystin-LR (MC-LR) is a growing concern. In this study, we assessed the estrogenic potency of MC-LR in black-spotted frogs through combined field and laboratory approaches. In 13 bloom areas of Zhejiang province, China, the MC-LR concentrations in water ranged from 0.87 to 8.77 µg/L and were correlated with sex hormone profiles in frogs, suggesting possible estrogenic activity of MC-LR. Tadpoles exposed to 1 µg/L, an environmentally relevant concentration, displayed a female-biased sex ratio relative to controls. Transcriptomic results revealed that MC-LR induces numerous and complex effects on gene expression across multiple endocrine axes. In addition, exposure of male adults significantly increased the estradiol (E2)/testosterone (T) ratio by 3.5-fold relative to controls. Downregulation of genes related to male reproductive endocrine function was also identified. We also showed how MC-LR enhances the expression of specific estrogen receptor (ER) proteins, which induce estrogenic effects by activating the ER pathway and hypothalamic-pituitary-gonadal (HPG) axis. In aggregate, our results reveal multiple lines of evidence demonstrating that, for amphibians, MC-LR is an estrogenic endocrine disruptor at environmentally relevant concentrations. The data presented here support the need for a shift in the MC-LR risk assessment. While hepatoxicity has historically been the focus of MC-LR risk assessments, our data clearly demonstrate that estrogenicity is a major mode of toxicity at environmental levels and that estrogenic effects should be considered for risk assessments on MC-LR going forward.

Chronic Exposure to a PFAS Mixture Resembling AFFF-Impacted Surface Water Decreases Body Size in Northern Leopard Frogs (Rana pipiens).

Per- and polyfluoroalkyl substances (PFAS) occur in the environment as mixtures, yet mixture toxicity remains poorly understood. Aqueous film-forming foams (AFFFs) are a common source of PFAS. Our objective was to examine chronic effects of a complex PFAS mixture on amphibian growth and development. We tested toxicity of a five-chemical PFAS mixture summing to 10 µg/L and that accounts for >90% of the PFAS in AFFF-affected surface waters: perfluorooctane sulfonate (PFOS, 40%), perfluorohexane sulfonic acid (PFHxS, 30%), perflurooctanoic acid (PFOA, 12.5%), perfluorohexanoic acid (PFHxA, 12.5%), and perfluoropentanoic acid (PFPeA, 5%). We also included treatments to determine whether PFOS drove mixture toxicity and whether PFOS and mixture components act additively. We exposed Northern leopard frog (Rana pipiens) larvae through metamorphosis (∼130 d) in outdoor mesocosms. After 21 days of exposure, the larval body condition fell ∼5% relative to controls in the 4 µg/L PFOS treatment and mixtures lacking PFOS. At metamorphosis, the full 5-component 10 µg/L PFAS mixture reduced mass by 16% relative to controls. We did not observe effects on development. Our results indicate that toxicity of PFOS and other PFAS mixtures typical of AFFF sites act additively and that PFOS is not more inherently toxic than other mixture components.

Toxicities of Legacy and Current-Use PFAS in an Anuran: Do Larval Exposures Influence Responses to a Terrestrial Pathogen Challenge?

Legacy polyfluoroalkyl substances (PFAS) [perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA)] are being replaced by various other fluorinated compounds, such as hexafluoropropylene oxide dimer acid (GenX). These alternatives are thought to be less bioaccumulative and, therefore, less toxic than legacy PFAS. Contaminant exposures occur concurrently with exposure to natural stressors, including the fungal pathogen Batrachocytrium dendrobatidis (Bd). Despite evidence that other pollutants can increase the adverse effects of Bd on anurans, no studies have examined the interactive effects of Bd and PFAS. This study tested the growth and developmental effects of PFOS, PFOA, and GenX on gray treefrog (Hyla versicolor) tadpoles, followed by a Bd challenge after metamorphosis. Despite PFAS exposure only occurring during the larval stage, carry-over effects on growth were observed post metamorphosis. Further, PFAS interacted with Bd exposure to influence growth; Bd-exposed animals had significantly shorter SVL [snout-vent length (mm)] with significantly increased body condition, among other time-dependent effects. Our data suggest that larval exposure to PFAS can continue to impact growth in the juvenile stage after exposure has ended. Contrary to predictions, GenX affected terrestrial performance more consistently than its legacy congener, PFOA. Given the role of Bd in amphibian declines, further investigation of interactions of PFAS with Bd and other environmentally relevant pathogens is warranted.

Does salinity mediate the toxicity of perfluorooctanesulfonic acid (PFOS) in an estuarine fish?

Perfluorooctanesulfonic acid (PFOS) is detected in estuarine environments, where salinity levels fluctuate regularly. We investigated the effects of salinity on the toxicity of PFOS in embryos and larvae of Cyprinodon variegatus. We crossed six PFOS treatments (0, 1-10,000 µg/L) with two salinities (10, 30 ppt). Larvae exposed to the highest concentration of PFOS under high salinity accumulated over twice the amount of PFOS compared to larvae maintained under low salinity. Embryonic survival was unaffected by PFOS, salinity, or their interaction. PFOS delayed time to hatch and increased salinity reduced time to hatch regardless of PFOS treatment; however, no salinity by PFOS interactions were observed. Conversely, PFOS and salinity interacted in the larval stage, with decreased survival at 30 ppt salinity. This is one of the first studies evaluating interactive effects of PFOS and high salinity and highlights the importance of assessing PFAS toxicity across life stages.

Occurrence and implications of per and polyfluoroalkyl substances in animal feeds used in laboratory toxicity testing.

The exceptional thermal and chemical stability and the amphiphilicity of per- and polyfluoroalkyl substances (PFAS) have resulted in widespread use and subsequent contamination in environmental media and biota. Concerns surrounding toxicity have led to numerous animal-based toxicity studies. Due to the ubiquity of PFAS and the low parts per trillion (ppt) health advisory levels for drinking water, several contamination elimination protocols have been implemented. In addition, it is urgently necessary to perform low-dose experiments, but due to unknown pathways for entry of unwanted PFAS, low-dose studies are extremely challenging to conduct. However, animal feed sources are a likely route that could introduce unwanted PFAS into experiments, yet investigations of PFAS in common animal feeds are lacking. Here, we report the examination of PFAS levels in eighteen different animal feeds, representing a range of diets fed to diverse taxa. We evaluated whether PFAS levels in feeds were correlated with ingredient composition (plant versus animal-based) or dietary habits of lab animals (amphibian, fish, invertebrate, mammal). PFOS, PFHxS, PFOA, and short-chain perfluoroalkyl carboxylic acids had the highest detection levels and frequencies across all samples. Different food ingredients led to different PFAS profiles. No meaningful levels of PFAS precursors were detected. We demonstrate that PFAS contamination in animal feed is pervasive. Reducing food-sourced PFAS is a critical, albeit challenging task to improve interpretability of in vivo exposures.

An Environmentally Relevant Mixture of Perfluorooctanesulfonic Acid and Perfluorohexanesulfonic Acid Does Not Conform to Additivity in Northern Leopard Frogs Exposed Through Metamorphosis.

Per- and polyfluoroalkyl substances (PFAS) are chemicals associated with adverse health effects. At aqueous film-forming foam sites, they occur as mixtures, with perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) commonly co-occurring in the highest concentrations. Although PFOS and PFHxS toxicities have been studied, few studies have tested their potential interaction. Using Rana pipiens, the present study compared toxicities of a 1:1 PFOS:PFHxS mixture to PFOS and PFHxS individually with the prediction that responses would be additive. Gosner stage 25 (GS 25) tadpoles were exposed through metamorphosis (GS 46) to 0.5 and 1 ppb PFOS or PFHxS alone or to a mixture of 0.5 ppb PFOS and 0.5 ppb PFHxS. Tadpoles were weighed and measured (snout-vent length [SVL]) at day 31, metamorphic climax (GS 42), and GS 46. These values were used to calculate the scaled mass index (SMI), a measure of body condition. Body burdens were quantified on day 31 and at GS 46. The PFOS and PFHxS body burdens were elevated relative to controls at GS 46. No effects were observed on survival, SVL, or mass. Single PFAS effects included a 17% reduction in SMI at day 31 (0.5 ppb PFHxS) and a 1.1-day longer metamorphic period (1 ppb PFHxS) relative to controls. Mixture results deviated from additivity-SMIs were higher than expected on day 31 and lower than expected at GS 42. In addition, time to GS 42 in the PFAS mixture exceeded expected additivity by 12 days. Results from a chronic exposure to a 1:1 PFOS:PFHxS mixture resulted in changes in body condition and length of metamorphosis that deviated from additivity. More PFAS mixture toxicity studies conducted at relevant ratios and concentrations are needed. Environ Toxicol Chem 2022;41:3007-3016. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Dietary exposure and accumulation of per- and polyfluoroalkyl substances alters growth and reduces body condition of post-metamorphic salamanders.

Per- and polyfluoroalkyl substances (PFAS) are contaminants of concern due to their persistence, potential to bioaccumulate, and toxicity. While dietary exposure is the primary route of exposure for terrestrial species, data on dietary PFAS uptake and adverse effects are largely restricted to mammals. As such, substantial data gaps exist that hinder ecological risk assessment, including environmentally relevant exposure levels and taxa. Using a 30-d laboratory experiment, we examined the effects of dietary PFAS-exposure on post-metamorphic tiger salamanders (Ambystoma tigrinum). We fed salamanders crickets exposed to perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), or 6:2 fluorotelomer sulfonate (6:2 FTS) at low (<1.0), medium (2-5), or high (16-62) ng PFAS/g/d (wet weight) dose rates. We found that only PFOS resulted in substantial biomagnification. Despite limited evidence for biomagnification, PFAS altered growth and generally reduced body condition. Salamanders with the highest burdens of PFOS grew less and had lower body conditions, while burdens of PFHxS and PFOA were only associated with reduced growth. There was no evidence that environmentally relevant doses of PFAS increase liver size in salamanders. Our results demonstrate that dietary exposure and accumulation of PFAS can impact fitness-related traits in amphibians and contribute to trophic transfer in terrestrial food webs.

Effects of larval atrazine exposure in mesocosms on Blanchard's cricket frogs (Acris blanchardi) reared through overwintering and to reproductive age.

We exposed Blanchard's cricket frog (Acris blanchardi) tadpoles to atrazine in simulated aquatic communities (outdoor mesocosms) at nominal concentrations of 0, 1, 10, 100, and 200 µg/L and tracked the effects of exposure to spring emergence in the laboratory, as well as to reproductive age in outdoor, terrestrial enclosures. We tested hypotheses that 1) atrazine addition increases the prevalence and intensity of testicular ova (TO) among phenotypic males at metamorphosis and after overwintering, 2) atrazine reduces maturation of ova after overwintering among phenotypic females, and 3) atrazine alters mass, time, and survival to metamorphosis, as well as growth and survival across terrestrial life stages. Atrazine addition increased probability of TO presence at metamorphosis, but only when treatments were pooled and compared to the control, where background atrazine was detected. Atrazine did not influence the intensity of TO among metamorphs. We observed TO among males at spring emergence and at reproductive age regardless of exposure concentration. We found no evidence for effects of exposure on gonadal maturation among females after overwintering. Exposure to 200 µg/L reduced survival to metamorphosis, but atrazine did not affect mass at metamorphosis, time to metamorphosis, or survival or mass after overwintering. We demonstrate that atrazine addition can increase TO prevalence relative to background rates at metamorphosis and that TO are also present among phenotypic males after overwintering. We suggest that this non-model species is sensitive to effects of larval EDC exposures on gonadal development and morphology and that further work with cricket frogs is warranted.

Atrazine feminizes sex ratio in Blanchard's cricket frogs (Acris blanchardi) at concentrations as low as 0.1 µg/L.

We exposed Blanchard's cricket frogs (Acris blanchardi) to ecologically relevant concentrations (0, 0.1, 1, and 10 µg/L) of a commercial formulation of atrazine throughout the larval period to determine effects on survival, somatic growth and development (time to metamorphosis and mass at metamorphosis), and gonadal development (sex ratio at metamorphosis and the prevalence of testicular ova in phenotypic males). We tested the following hypotheses: 1) atrazine feminizes the sex ratio, 2) atrazine increases the proportion of phenotypic males with testicular ova, and 3) atrazine differentially affects somatic growth (mass at metamorphosis) and development (time to metamorphosis) for males and females. Although the control sex ratio was male-biased, exposure to 0.1 and 10 µg/L atrazine feminized sex ratios, because these treatments produced 51 and 55% fewer males than the control, respectively. We did not observe testicular ova. Atrazine did not impact survival or metamorphosis, and we did not detect sexually dimorphic impacts on time to metamorphosis or mass at metamorphosis. However, males metamorphosed 2.3 d later than females, regardless of treatment. Sex biases in timing of metamorphosis are underexplored in anurans, but if prevalent, could have important implications for theory surrounding the impact of environmental factors on metamorphosis. Our data suggest that cricket frog sex ratios are sensitive to environmentally relevant concentrations of atrazine and that feminization in the field is likely. Environ Toxicol Chem 2018;37:427-435. © 2017 SETAC.

Variation in malathion sensitivity among populations of Blanchard's cricket frogs (Acris blanchardi) and implications for risk assessment.

Intraspecific variability in contaminant sensitivity could undermine risk assessments for nontarget organisms such as amphibians. To test how amphibian populations vary in tolerance to anticipated lethal and sublethal exposures to a pesticide, we exposed Blanchard's cricket frogs (Acris blanchardi) from 3 populations across a broad portion of their range to the insecticide malathion. Exposure in mesocosms to a nominal concentration of 1 mg/L (measured concentrations at 1 h and 24 h postaddition of 0.160 mg/L and 0.062 mg/L, respectively), a realistic direct-overspray scenario, reduced survival to metamorphosis by 43% relative to controls and revealed variation in tolerance among populations. Survival ranged from 74% for the most tolerant population to 18% for the least tolerant population, a 4.1-fold difference. Mass at metamorphosis and time to metamorphosis were unaffected. Although malathion reduced zooplankton abundance, it did not alter food resources (periphyton or phytoplankton relative abundance), or a suite of water-quality variables (pH, temperature, and dissolved oxygen). A 96-h time-to-death assay designed to isolate direct, lethal effects also revealed variation in tolerance among populations. Time to death (mean ± standard error) ranged from 2.4 ± 0.18 h for the least tolerant population to 17.8 ± 4.72 h for the most tolerant population, a 7.4-fold difference. However, relative sensitivities of populations differed in the mesocosm and laboratory studies, which differed in exposure concentrations, suggesting that populations tolerant of high concentrations can be more sensitive to lower concentrations. We suggest that direct overspray could reduce larval survival in the field for this species. Studies assessing the role of contaminants in declines or extrapolating to untested populations, especially across large geographical regions, should quantify the range of intraspecific variation. Risk assessors could address intraspecific variability directly by using an intraspecific uncertainty factor. Environ Toxicol Chem 2017;36:1917-1923. © 2016 SETAC.

Short-term atrazine exposure at breeding has no impact on Blanchard's cricket frog (Acris blanchardi) reproductive success.

Studies of endocrine-disrupting contaminants have focused on early-life exposures, but later exposures could impact fitness. We exposed adult frogs (Acris blanchardi) at reproduction to ecologically relevant atrazine concentrations (0, 1, or 10 µg/L) in outdoor arenas. We measured likelihood of breeding and number of resulting tadpoles. Atrazine impacted neither the probability of breeding nor the number of tadpoles produced, suggesting anuran reproductive success may not be impacted by short-term exposure to low concentrations. Environ Toxicol Chem 2017;36:3284-3288. © 2017 SETAC.

Effectiveness of a Low-Cost, Graduate Student-Led Intervention on Study Habits and Performance in Introductory Biology.

Institutions have developed diverse approaches that vary in effectiveness and cost to improve student performance in introductory science, technology, engineering, and mathematics courses. We developed a low-cost, graduate student-led, metacognition-based study skills course taught in conjunction with the introductory biology series at Miami University. Our approach aimed to improve performance for underachieving students by combining an existing framework for the process of learning (the study cycle) with concrete tools (outlines and concept maps) that have been shown to encourage deep understanding. To assess the effectiveness of our efforts, we asked 1) how effective our voluntary recruitment model was at enrolling the target cohort, 2) how the course impacted performance on lecture exams, 3) how the course impacted study habits and techniques, and 4) whether there are particular study habits or techniques that are associated with large improvements on exam scores. Voluntary recruitment attracted only 11-17% of our target cohort. While focal students improved on lecture exams relative to their peers who did not enroll, gains were relatively modest, and not all students improved. Further, although students across both semesters of our study reported improved study habits (based on pre and post surveys) and on outlines and concept maps (based on retrospectively scored assignments), gains were more dramatic in the Fall semester. Multivariate models revealed that, while changes in study habits and in the quality of outlines and concept maps were weakly associated with change in performance on lecture exams, relationships were only significant in the Fall semester and were sometimes counterintuitive. Although benefits of the course were offset somewhat by the inefficiency of voluntary recruitment, we demonstrate the effectiveness our course, which is inexpensive to implement and has advantage of providing pedagogical experience to future educators.